CN104531867A - Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit - Google Patents
Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit Download PDFInfo
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to a fluorescent quantitative PCR rapid detection kit for specifically detecting clostridium perfringens enterotoxin positive bacteria infection. The detection kit comprises (a) a fluorescent quantitative reaction solution; (b) clostridium perfringens exotoxin cpa positive and clostridium perfringens enterotoxin cpe positive quality control substances; and (c) a negative quality control substance, wherein the fluorescent quantitative reaction solution comprises 2 pairs of primers and one pair of probes, namely a forward primer cpa01 and a reverse primer cpa02 of clostridium perfringens exotoxin cpa and a fluorescence probe cpap, as well as a forward primer cpe01 and a reverse primer cpe02 of clostridium perfringens enterotoxin cpe and a fluorescence probe cpep. The detection kit disclosed by the invention has the advantages that positive clostridium perfringens of the enterotoxin cpe can be rapidly diagnosed; the specificity is high, the sensitivity is high, and the false positive rate is low; and moreover, the detection speed is high, and only one and a half hours are needed.
Description
Technical field
The present invention relates to the fluorescent quantificationally PCR detecting kit of a species specificdetection clostridium perfringens enterotoxin positive bacterial infection, belong to bacterial nucleic acid detection field, be applicable to the fast qualitative detection by quantitative to the clostridium perfringens enterotoxin positive in clinical and scientific research.
Background technology
Clostridium perfringens (Clostridium perfringens) is a kind of pathogenic agent of important zoonosis, is extensively present in the soil in environment, water source, humans and animals enteron aisle.The pathogenic Main Basis of this bacterium its produce the ability of various toxin, up to the present, find the clostridium perfringens toxoid of more than at least 15 kinds altogether, except four kinds of somatotype lethal toxins α, β, ε, ι toxin of somatotype, wherein the clostridium perfringens of all models is all containing extracellular toxin cp α, also find the important new bacteria toxin having substantial connection with humans and animals disease, as enterotoxin (clostridium perfringens enterotoxin, CPE).Although enterotoxigenic clostridium perfringens accounts for 2 ~ 5% of clostridium perfringens, but the clostridium perfringens of the CPE positive can cause food poisoning, non-food source property gastrointestinal illness and the amimal gastroenteropathy such as antibiotic associated diarrhea and sporadic diarrhoea of people, closely bound up with food public safety, at present according to international newest standards, the index that in sample, CPE positive clostridium perfringens is poisoned by food as clostridium perfringens will be detected.
Existing panimmunity method for detecting the enterotoxin in clinical sample, but has the limitation of detection, and what immunological detection method detected is toxin protein, easily occurs false-negative detected result.Sample such as the enterotoxin content such as diarrheic stools mainly owing to detecting are in lower level, on the other hand because enterotoxin results from the gemma stage of this bacterium, when cultivating in vitro, be difficult to form gemma and produce enterotoxin albumen, therefore immunological detection method conveniently can not detect from artificial culture thing.Along with the widespread use of round pcr, from traditional method, rapid detection modernism based on PCR and hybridization probe is transitioned into the detection of clostridium perfringens, establish multi-PCR detection method according to the different toxin of clostridium perfringens at present, can detect and somatotype clostridium perfringens.Although PCR method sensitivity is higher, high specificity, also have some defects, can not detect the clinical sample of extremely low viral level, the specificity how improving method still needs further to be studied.
Fluorescence quantitative PCR detection technique is a high again new level on the basis of regular-PCR, and susceptibility, specificity and detection speed all have significant advantage.But it it is also proposed higher requirement to primer and probe.Clostridium perfringens enterotoxin cpe has two kinds of genotype, and one is positioned on bacterial chromosome, and one is on bacterial plasmid.
Prior art is little about the document of clostridium perfringens antigen, antibody test and fluorescent quantitative PCR technique, does not relate to the diagnostic techniques of clostridium perfringens extracellular toxin cpa and enterotoxin cpe.
Summary of the invention
The object of the invention is to set up a kind of fluorescent quantitative PCR technique specific detection that utilizes containing the quantitative fluorescent PCR quick detection kit of enterotoxin cpe clostridium perfringens, this test kit specially can detect clostridium perfringens enterotoxin cpe positive bacteria delicately, thus reaches the object of quick diagnosis.
The present invention is the technical scheme solving the employing of above technical problem: clostridium perfringens enterotoxin positive bacteria double fluorescent quantitative PCR quick detection kit, is characterized in that this test kit comprises: a) fluorescent quantitation reaction solution, b) the clostridium perfringens extracellular toxin cpa positive and clostridium perfringens enterotoxin cpe positive quality control product, c) negative quality control product; Wherein fluorescent quantitation reaction solution includes primer 2 to right with probe 1, is upstream primer cpa01 and the downstream primer cpa02 and fluorescent probe cpap of clostridium perfringens extracellular toxin cpa; The upstream primer cpe01 of clostridium perfringens enterotoxin cpe and downstream primer cpe02 and fluorescent probe cpep, wherein,
The primer sequence of clostridium perfringens extracellular toxin cpa is:
Upstream primer cpa01:5 '-GATTTGTAAGGCGCTTATTTGT-3 ',
Downstream primer cpa02:5 '-ATAGCATGAGTTCCTGTTCCA-3 ',
Fluorescent probe cpap:5 '-CTACGCTAGCAACTAGCCTATGGGCTG-3 ', the fluorescent reporter gene of probe upstream 5 ' end mark is TAMRA, and the fluorescent quenching group of 3 ' end mark is BHQ-2;
The primer sequence of clostridium perfringens enterotoxin cpe is:
Upstream primer cpe01:5 '-AGTGTAAATTAAGCTTTTGAGTCCA-3 ',
Downstream primer cpe02:5 '-TAGCTGCTGCTACAGAAAGATTAA-3 ',
Fluorescent probe cpep is 5 '-AAGGGTATGAGTTAGAAGAACGCCAATCA-3 ', and the fluorescent reporter gene of probe upstream 5 ' end mark is FAM, and the fluorescent quenching group of 3 ' end mark is BHQ-1.
By such scheme, described fluorescent quantitation reaction solution by 1 × PCR buffer (containing Mg
2+), dNTPs 0.5mM, Taq enzyme 2U, each 0.4 μM of the upstream primer cpa01 of clostridium perfringens extracellular toxin cpa and downstream primer cpa02, fluorescent probe cpap is 0.4 μM, each 0.4 μM of the upstream primer cpe01 of clostridium perfringens enterotoxin cpe and downstream primer cpe02, fluorescent probe cpep are 0.4 μM of composition.
By such scheme, the described clostridium perfringens extracellular toxin cpa positive and clostridium perfringens enterotoxin cpe positive quality control product are the cloned plasmids containing alpha toxin and cpe sequence, wherein the positive quality control product of clostridium perfringens extracellular toxin cpa is the carrier pUC57-cpa built, and its sequence is: ATGAA AAGAA AGATT TGTAA GGCGC TTATT TGTGC TACGC TAGCA ACTAGCCTAT GGGCT GGGGC ATCAA CTAAA GTCTA CGCTT GGGAT GGAAA GATTG ATGGA ACAGG AACTC ATGCTATGAT TG;
Clostridium perfringens enterotoxin cpe positive quality control product is the carrier pUC57-cpe that the long-chain of synthesis builds, and its sequence is AGATATAGAA AAAGA AATCC TTGAT TTAGC TGCTG CTACA GAAAG ATTAA ATTTA ACTGA TGCAT TAAAC TCAAATCCAG CTGGT AATTT ATATG ATTGG CGTTC TTCTA ACTCA TACCC TTGGA CTCAA AAGCT TAATT TACACTTAAC AATTA CAGCT ACTGG ACAAA AATAT AGAAT CTTAG CTAGC AAAAT TGTTG ATTTT AATAT TTATTCAAAT AATTT TAATA ATCTA GTGAA ATT.
By such scheme, described negative quality control product be sterilizing without enzyme distilled water.
The method that the present invention sets up utilizes fluorescent quantitation technology, design the fluorescent label DNA probe that two have high specific, by initial point quantitatively and fluorescence detecting system Real-Time Monitoring accumulation fluorescence intensity and realize exotoxin gene cpa and enterotoxin genes cpe that clostridium perfringens detected.Directly verify whether this bacterium is the positive clostridium perfringens of enterotoxin, overcomes the limitation of ordinary method by single test result.
The present invention compared with prior art has the following advantages:
1, quick diagnosis can be carried out to the clostridium perfringens of the enterotoxin cpe positive, effective examination can be carried out to the clostridium perfringens enterotoxin positive bacteria of harm public food safety, compensate for the vacancy of existing detection technique;
2, specificity is good, highly sensitive, and false positive rate is low.By the high specific hybridization two ore control of the amplification of the two pairs of Auele Specific Primer high specifics and two fluorescent probes, there is very high accuracy; Reaction process is monitored in real time by fluorescence detecting system, and fluorescence detecting system has very high detection sensitivity to fluorescent signal;
3, detection speed is fast, only needs 1 and a half hours.There is no post-processed, need not hybridize, electrophoresis, the process such as to take pictures, all reagent is all once increase, and does not need to uncap, and does not produce pollution.
Accompanying drawing explanation
Fig. 1 is the amplification curve that extracellular toxin cpa standard substance pUC57-cpa 10 times of gradient dilutions carry out quantitative fluorescent PCR and obtain, X-coordinate represents cycle number, ordinate zou represents fluorescence intensity, in figure, the straight line of parallel principle X-coordinate represents fluorescence threshold values, 1. ~ be 6. followed successively by 10 times of extracellular toxin cpa standard substance diluted;
Fig. 2 is the typical curve carrying out made by fluorescent quantitation result according to extracellular toxin cpa standard substance pUC57-cpa 10 times of dilutions, and X-coordinate represents extracellular toxin cpa standard substance extension rate logarithmic value, and ordinate zou represents cycle number;
Fig. 3 is the amplification curve that enterotoxin cpe standard substance pUC57-cpe 10 times of dilutions are carried out quantitative fluorescent PCR and obtained, X-coordinate represents cycle number, ordinate zou represents fluorescence intensity, in figure, the straight line of parallel principle X-coordinate represents fluorescence threshold values, 1. ~ be 5. followed successively by 10 times of enterotoxin cpe standard substance diluted;
Fig. 4 is the typical curve carrying out made by fluorescent quantitation result according to enterotoxin cpe standard substance pUC57-cpe 10 times of dilutions, and X-coordinate represents enterotoxin cpe standard substance extension rate logarithmic value, and ordinate zou represents cycle number.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be appreciated that these examples are only not used in restriction the scope of protection of present invention for illustration of the present invention.
Embodiment 1
The positive double fluorescent quantitative PCR quick detection kit composition of clostridium perfringens enterotoxin and preparation
1. reagent composition:
EasyTaq enzyme (5U/ μ L), dNTPs (10mM), all purchased from Promega company; PCR primer pair cpa01, cpa02 and cpe01, cpe02 and probe cpap, cpep synthesize by Shanghai Sheng Gong bio-engineering corporation;
2. preparation of reagents
A) fluorescent quantitation reaction solution: 1 × PCR Buffer is (containing Mg
2+), dNTPs 0.5mM, Taq enzyme 2U, each 0.4 μM of the upstream primer cpa01 of clostridium perfringens extracellular toxin cpa and downstream primer cpa02, fluorescent probe cpap is 0.4 μM, each 0.4 μM of the upstream primer cpe01 of clostridium perfringens enterotoxin cpe and downstream primer cpe02, fluorescent probe cpep is 0.4 μM.The wherein primer sequence of extracellular toxin cpa, upstream primer cpa01:5 '-GATTTGTAAGGCGCTTATTTGT-3 ', downstream primer cpa02:5 '-ATAGCATGAGTTCCTGTTCCA-3 ', probe primer cpap:5 '-CTACGCTAGCAACTAGCCTATGGGCTG-3 '.The fluorescent reporter gene of probe upstream 5 ' end mark is TAMRA, and the fluorescent quenching group of 3 ' end mark is BHQ-2; Enterotoxin cpe primer sequence is, upstream primer cpe01:5 '-AGTGTAAATTAAGCTTTTGAGTCCA-3 ', downstream primer cpe02:5 '-TAGCTGCTGCTACAGAAAGATTAA-3 ', probe primer cpep is 5 '-AAGGGTATGAGTTAGAAGAACGCCAATCA-3 ', the fluorescent reporter gene of probe upstream 5 ' end mark is FAM, and the fluorescent quenching group of 3 ' end mark is BHQ-1;
B) the clostridium perfringens extracellular toxin cpa positive and enterotoxin cpe positive quality control product are the cloned plasmids containing alpha toxin and cpe sequence: wherein the positive quality control product of extracellular toxin cpa is the carrier pUC57-cpa built, and its sequence is: ATGAA AAGAA AGATTTGTAA GGCGC TTATT TGTGC TACGC TAGCA ACTAG CCTAT GGGCT GGGGC ATCAA CTAAA GTCTA CGCTTGGGAT GGAAA GATTG ATGGA ACAGG AACTC ATGCT ATGAT TG;
Enterotoxin cpe positive quality control product is the carrier pUC57-cpe that the long-chain of synthesis builds, and its sequence is AGATA TAGAA AAAGAAATCC TTGAT TTAGC TGCTG CTACA GAAAG ATTAA ATTTA ACTGA TGCAT TAAAC TCAAA TCCAG CTGGTAATTT ATATG ATTGG CGTTC TTCTA ACTCA TACCC TTGGA CTCAA AAGCT TAATT TACAC TTAAC AATTACAGCT ACTGG ACAAA AATAT AGAAT CTTAG CTAGC AAAAT TGTTG ATTTT AATAT TTATT CAAAT AATTTTAATA ATCTA GTGAA ATT.
C) negative quality control product: sterilizing without enzyme distilled water.
Embodiment 2
The using method of the positive double fluorescent quantitative PCR rapid detection primer of clostridium perfringens enterotoxin and test kit
1, the process of sample
Tested sample is tissue or liquid sample: by national standard method, sterile sampling product 10g (mL) puts in 90mL 0.5% buffered peptone water, and jolting mixes, and then sample thief liquid is inoculated in TSC substratum and carries out increasing bacterium, 37 DEG C of Anaerobic culturel 24h.
2, the process of tested sample adopts the genomic dna of quick boiling method extracting bacterium, as the template of quantitative fluorescent PCR reaction.Single bacterium colony on picking TSC substratum, puts into containing 200 μ l sterilizing deionized waters, heated and boiled 15min, and take out test tube, the centrifugal 5min of 10000rpm, gets the template that supernatant reacts as quantitative fluorescent PCR.
3, quantitative fluorescent PCR reaction
Get each 18 μ l of fluorescent quantitation reaction solution respectively, get each 2 μ l of template of the 2nd step gained, add different PCR reaction tubess respectively, carry out PCR reaction with fluorescent quantitative detector and the fluorescence intensity discharged in real-time detection reaction process, PCR is obtained by reacting clostridium perfringens extracellular toxin cpa specific amplification goal gene sequence and is: GATTT GTAAG GCGCT TATTT GTGCT ACGCT AGCAA CTAGC CTATG GGCTG GGGCA TCAAC TAAAG TCTAC GCTTG GGATG GAAAG ATTGA TGGAA CAGGA ACTCA TGCT.The clostridium perfringens enterotoxin cpe positive bacteria specific amplification goal gene sequence that PCR is obtained by reacting is: AGTGT AAATT AAGCT TTTGA GTCCA AGGGT ATGAG TTAGA AGAAC GCCAA TCATA TAAAT TACCA GCTGG ATTTG AGTTT AATGC ATCAG TTAAA TTTAA TCTTT CTGTA GCAGC AGC.Select fluorescent signal FAM and TAMRA.Cycling condition is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s
56 DEG C of annealing, extend 30s, 40 circulations of increasing.The program setting of fluoroscopic examination is carried out double fluorescent detection at the end of each circulation second, and determined wavelength is 530nm; Threshold value is set to the vertex of threshold line just above normal negative sample.
4, result judges
A) interpretation of result condition setting
After loop ends, instrument is used to carry software analysis detected result.Circulation thresholding (Threshold cycle, Ct) setting principle, according to the adjustment of noise of instrument situation, is as the criterion with the vertex of Ct value just above normal negative sample amplification curve;
B) quality control standard
Negative control is without Ct value and without amplification curve;
About 20, and typical amplification curve should be there is in positive control Ct value.Otherwise it is invalid that this time experiment is considered as.
Result judges
Negative findings judges: without Ct value, and without typical amplification curve;
Positive findings judges: Ct value is less than 35, and occurs typical amplification curve;
Embodiment 3
The application of the positive double fluorescent quantitative PCR rapid detection primer of clostridium perfringens enterotoxin and test kit
1, sensitivity test
With the extracellular toxin cpa positive quality control product pUC57-cpa (1.5 × 10 of 10 times of serial dilutions
4~ 1.5 × 10
-2and the positive quality control product pUC57-cpe (1.8 × 10 of enterotoxin cpe ng/ml)
3~ 1.8 × 10
-2ng/ml) as template, quantitative real time PCR Instrument detects, obtain real time PCR amplification curve and typical curve is shown in accompanying drawing 1, accompanying drawing 2, accompanying drawing 3, accompanying drawing 4 respectively.Shown by accompanying drawing 1, when positive quality control product plasmid concentration>=1.5 × 10 of extracellular toxin cpa
-2during ng/ml, kinetic curve is in rising trend, shown in accompanying drawing 3, when positive quality control product plasmid concentration>=1.8 × 10 of enterotoxin cpe
-2during ng/ml, kinetic curve is in rising trend.Therefore, the detection sensitivity of the extracellular toxin cpa of the method is 1.5 × 10
-2the detection sensitivity of ng/ml, enterotoxin cpe is 1.8 × 10
-2ng/ml.It is 1.5 × 10 that accompanying drawing 2 and accompanying drawing 4 show the quantitative linearity range of the method respectively
4~ 1.5 × 10
-2ng/ml and 1.8 × 10
3~ 1.8 × 10
-2ng/ml, coefficient R
2be respectively 0.991 and 0.996.
2, specific test
The positive double fluorescent quantitative PCR kit of clostridium perfringens enterotoxin is adopted to carry out specific test to intestinal bacteria O78, Escherichia coli O 157: H7, bacillus coli DH 5 alpha, Salmonellas, pasteurellosis bacillus, campylobacter jejuni, campylobacter coli, campylobacter coli, streptococcus aureus, swine streptococcus, faecalis, actinobacillus pleuropneumoniae, feminine gender is to the detected result of above-mentioned 12 kinds of bacteriums, detect the various tissues of water, chicken and duck in addition, result is feminine gender.This test kit of above result sufficient proof has good specificity in clinical sample detects.
Detect the local strains that A type and C type perfringens shuttle reference culture and other 30 strain laboratories are separated preservation voluntarily, detected result is the positive.It is good that preliminary proof the method detects adaptability to the different serotypes of clostridium perfringens.
3, replica test
Replica test between the clostridium perfringens sample group of getting 3 parts of different concns, obtain table 1 and table 2, calculate the repeated between-group variation coefficient β value (see table 1) detected of clostridium perfringens extracellular toxin cpa is respectively 4.53%, 3.18%, 2.52%, the between-group variation coefficient β value (see table 2) that enterotoxin cpe repeatability detects is respectively 3.9%, 4.6%, 2.6%, all be less than 5%, prove this detection method repeatability very well, there is good specificity.
Between the group of table 1 sample, repeatability detects the result of clostridium perfringens different concns extracellular toxin cpa
Between the group of table 2 sample, repeatability detects the result of clostridium perfringens different concns enterotoxin cpe
A: circulation thresholding
B: mean CT-number
Claims (4)
1. clostridium perfringens enterotoxin positive bacteria double fluorescent quantitative PCR quick detection kit, is characterized in that this test kit comprises: a) fluorescent quantitation reaction solution, b) the clostridium perfringens extracellular toxin cpa positive and clostridium perfringens enterotoxin cpe positive quality control product, c) negative quality control product; Wherein fluorescent quantitation reaction solution includes primer 2 to right with probe 1, is upstream primer cpa01 and the downstream primer cpa02 and fluorescent probe cpap of clostridium perfringens extracellular toxin cpa; The upstream primer cpe01 of clostridium perfringens enterotoxin cpe and downstream primer cpe02 and fluorescent probe cpep, wherein,
The primer sequence of clostridium perfringens extracellular toxin cpa is:
Upstream primer cpa01:5 '-GATTTGTAAGGCGCTTATTTGT-3 ',
Downstream primer cpa02:5 '-ATAGCATGAGTTCCTGTTCCA-3 ',
Fluorescent probe cpap:5 '-CTACGCTAGCAACTAGCCTATGGGCTG-3 ', the fluorescent reporter gene of probe upstream 5 ' end mark is TAMRA, and the fluorescent quenching group of 3 ' end mark is BHQ-2;
The primer sequence of clostridium perfringens enterotoxin cpe is:
Upstream primer cpe01:5 '-AGTGTAAATTAAGCTTTTGAGTCCA-3 ',
Downstream primer cpe02:5 '-TAGCTGCTGCTACAGAAAGATTAA-3 ',
Fluorescent probe cpep is 5 '-AAGGGTATGAGTTAGAAGAACGCCAATCA-3 ', and the fluorescent reporter gene of probe upstream 5 ' end mark is FAM, and the fluorescent quenching group of 3 ' end mark is BHQ-1.
2., by clostridium perfringens enterotoxin positive bacteria double fluorescent quantitative PCR quick detection kit according to claim 1, it is characterized in that described fluorescent quantitation reaction solution by 1 × PCR buffer (containing Mg
2+), dNTPs 0.5mM, Taq enzyme 2U, each 0.4 μM of the upstream primer cpa01 of clostridium perfringens extracellular toxin cpa and downstream primer cpa02, fluorescent probe cpap is 0.4 μM, each 0.4 μM of the upstream primer cpe01 of clostridium perfringens enterotoxin cpe and downstream primer cpe02, fluorescent probe cpep are 0.4 μM of composition.
3. by clostridium perfringens enterotoxin positive bacteria double fluorescent quantitative PCR quick detection kit according to claim 1, it is characterized in that the described clostridium perfringens extracellular toxin cpa positive and clostridium perfringens enterotoxin cpe positive quality control product are the cloned plasmids containing alpha toxin and cpe sequence, wherein the positive quality control product of clostridium perfringens extracellular toxin cpa is the carrier pUC57-cpa built, its sequence is: ATGAA AAGAA AGATT TGTAA GGCGC TTATT TGTGC TACGC TAGCA ACTAG CCTATGGGCT GGGGC ATCAA CTAAA GTCTA CGCTT GGGAT GGAAA GATTG ATGGA ACAGG AACTC ATGCT ATGATTG,
Clostridium perfringens enterotoxin cpe positive quality control product is the carrier pUC57-cpe that the long-chain of synthesis builds, and its sequence is AGATATAGAA AAAGA AATCC TTGAT TTAGC TGCTG CTACA GAAAG ATTAA ATTTA ACTGA TGCAT TAAAC TCAAATCCAG CTGGT AATTT ATATG ATTGG CGTTC TTCTA ACTCA TACCC TTGGA CTCAA AAGCT TAATT TACACTTAAC AATTA CAGCT ACTGG ACAAA AATAT AGAAT CTTAG CTAGC AAAAT TGTTG ATTTT AATAT TTATTCAAAT AATTT TAATA ATCTA GTGAA ATT.
4. by clostridium perfringens enterotoxin positive bacteria double fluorescent quantitative PCR quick detection kit according to claim 1, it is characterized in that described negative quality control product be sterilizing without enzyme distilled water.
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